PROJECT SUMMARY/ABSTRACT Human immunodeficiency virus-1 (HIV-1) can be managed with anti-retroviral therapies (ART), but a cure remains elusive. This is due to a reservoir of latently infected cells, which are established early in infection. One of the main viral reservoirs in the blood are memory CD4+ T cells. Latently infected cells harbor the virus yet are invisible to our immune system. Currently there is no distinctive latently infected HIV-1 cell marker, so straightforward targeting or identification of these latent cells is not yet possible. One current strategy toward an HIV cure involves flushing the latent virus out of hiding using drugs or small molecules called latency reversing agents (LRAs). Once the latent virus is induced, the latent cell can be eliminated by viral cytopathicity or immune recognition. HIV is incredibly genetically diverse and there are at least 9 subtypes worldwide, in addition to an ever-increasing number of recombinant forms. Most of our knowledge of HIV latency comes from studies utilizing subtype B viruses. There is mounting in vitro and in vivo evidence that suggests that there are subtype-specific differences. Specifically, subtype-dependent differences in disease progression, viral replication, viral protein function, and responsiveness to cytokine have been observed. There is a paucity of knowledge on whether there are subtype-dependent differences in the establishment of latency. We hypothesize that non-subtype B viruses will establish latency at a lower frequency compared to subtype B and are differentially sensitive to LRAs due to subtype-specific factors. Our preliminary data suggests that in an in vitro primary cell model, subtype C establishes latency at a lower frequency than a previously characterized subtype B virus. We propose to study the potential relationship between viral subtype and latency using subtypes A, B, C, D, AE, AG, which covers approximately 85% of infections worldwide, in an in vitro central memory CD4 T cell model of latency. We will assess differences in latency by flow cytometry, digital droplet PCR, and Alu-PCR. We will examine the efficacy of several modern LRAs by flow cytometry and determine whether any differences observed are due to methylation or integration site. The work described in this application is critical to develop HIV cure strategies that could be applied worldwide.